Peroxidase Lab Report
Determining the Catalytic Properties of the Enzyme Peroxidase Extracted from a Turnip Under the Conditions of Temperature, pH, Boiling and Competitive Inhibitors By Robin Caserta BIO 101 September 30, 2013 ABSTRACT The enzyme, peroxidase, extracted from a turnip was tested for its efficiency in binding to its substrate and its stability under several conditions. To do this, we tested effects on peroxidase activity, first, with different amounts of the enzyme, next at temperatures of 4oC, Room Temperature, 32oC, 48oC and boiling; then, at pH 3, pH 5, pH 7 and pH 9; and, finally, with the competitive inhibitor, hydroxylamine.
We were able to measure enzymatic activity by the change in absorbance per second with a spectrophotometer. By testing different concentrations of peroxidase and its reaction rate in seconds, we were able to see that as the amount of enzyme increased the catalytic reaction also increased. The optimal amount of peroxidase concentration to be used in the subsequent experiments was determined to be 1. 0 mL. Any amount above this would have caused the rate of absorbance to be too fast, making it too difficult to get accurate readings. Any amount below this would not have produced a reaction “at an appreciable rate.
In addition, our results show that a rise temperature and pH only increase the rate of reaction to a certain point before the reaction rate begins to decline dramatically. In the case of boiling of the enzyme there was no rate of reaction found whatsoever. A similar result was found when hydroxylamine was added to the peroxidase and it caused an inhibition reaction. Overall, the results show that the peroxidase enzyme is sensitive with reference to the above factors in whether or not a reaction is catalyzed.
INTRODUCTION Enzymes are essential in the breakdown of certain materials or molecules that cannot be used by or are harmful to an organism as they are, into products that can be used or are not harmful for the organism. They are proteins and their structure consists of amino acids with a specific shape. Enzymes have an area called an active site where substrates (only a particular molecule or material to be converted) bind. When the substrate is bound to the active site on the whole entity becomes an enzyme-substrate complex.
The substrate’s covalent bond is disrupted and this chemical change constructs a new product from the original substrate while leaving the enzyme unaffected. Once this new product is released, the enzyme can bind again with more of these molecules needing conversion. Sometimes the enzyme works with coenzymes or cofactors such as vitamins or metallic ions to help the binding process. In other cases competitive inhibitors are at work and prevent a substrate from being bound to the active site on the enzyme.
The competitive inhibitor is similar enough to bind with the enzyme, but because it is not a perfect match, the enzyme then loses its ability to catalyze a reaction for that moment. In accordance with these properties, we will see how certain factors affect the reaction rate of peroxidase. For our purposes in this lab we used the enzyme peroxidase extracted from a turnip. Peroxidase, along with the help of its iron ion cofactor, catalyzes harmful hydrogen peroxide (H2O2) into a harmless compound and water.
In order to follow the rate of reaction for the breakdown of hydrogen peroxide, we used guaiacol, a colorless dye, which donates electrons and turns brown when it is oxidized. We used this dye so that we could measure the absorbance with the spectrophotometer as the hydrogen peroxide is being broken down and the color change gets stronger over specific time intervals. We developed several null hypotheses for these experiments: 1) The amount of enzyme added to the reaction will not affect the rate of reaction. emperature will not affect the enzymatic activity. 3) pH will not affect enzymatic activity. 4) Similar molecule to substrate will not affect enzymatic activity. MATERIALS AND METHODS Materials and methods are taken from Lab Topic 7 in the Biological Investigations, 9th Edition. RESULTS Graph 1- Effects of Peroxidase Amounts Graph 2 – Temperature Effects on Peroxidase Activity Graph 3 – pH Effects on Peroxidase Activity Graph 4 – Boiled Peroxidase Results Graph 5 – Hydroxylamine Results.
Optimum Temperature for Reaction Rate of Peroxidase Graph 7 – Optimum pH for Reaction Rate of Peroxidase In Graph 1, Effects of Peroxidase Amounts, it shows the difference in rates of reactions with different concentrations of peroxidase in the solution — Tubes 2 & 3 at 0. 5 ml, Tubes 4 & 5 at 1. 0 ml and Tubes 6 & 7 at 2. 0 ml, along with corresponding line slopes. Graph 2, Temperature Effects on Peroxidase Activity, shows the difference in rates of reaction for 1. 0 ml peroxidase at 4°C, Room Temperature ~ 23°C, 32°C and 48°C along with their corresponding line slopes.
This result allowed us to reject our hypothesis that the amount of enzyme added to the reaction will not affect the rate of reaction. This test was important so that we could ascertain the best amount of concentration to use in the subsequent experiments with the spectrophotometer set at absorbance 470 nm and timed recordings at 20-second intervals for a total of 2 minutes. At 0. 5 ml of peroxidase the reaction time was too slow thus no appreciable line or slope was rendered to measure the reaction with any accuracy.
Conversely, it was a challenge to get accurate absorbance readings at 2. 0 ml of peroxidase because the pace of the reaction appreciated so quickly and then met equilibrium. At 1. 0 ml of peroxidase the reaction time rendered an appreciable line and slope making it easier to record the absorbance every 20 seconds for 2 minutes and ultimately, the best concentration for use in the next experiments. It is known that when heat is applied to molecules, they move faster and collide more as the temperature rises.
This is also true for the enzyme peroxidase and its substrate until the temperature reaches 32°C and then the reaction begins to taper off and it dives down drastically at 48°C when the hydrogen bonds holding peroxidase structure together begin to break. The results of this test confirm the same by the slope of each line and thereby we are able to reject our hypothesis that temperature has no effect on peroxidase rate of reaction. At 4°C the slope of the line is 0. 0071, at Room Temperature ~ 23°C the slope is 0. 0094, at 32°C the slope is 0.
As for pH effects on peroxidase activity, Graph 3, indicates that the amount of acidity or basicness to a solution changes the three-dimensional structure of the enzyme and thereby changes the ability to bind with the substrate in an effective manner. Here we tested the null hypothesis: pH will not affect enzymatic activity. The results from Graph 3: pH Effects on Peroxidase Activity indicate that the more acidic pH 3 level disrupted the enzyme’s ability to bind with its substrate and its reaction rate did not appreciate noticeably.
As the solution became less acid at pH 5, the greatest reaction efficiency resulted. Once the pH was at 7 and beyond the reaction rate for peroxidase and its binding ability became poor and the reaction rate declined. Again, it was necessary to do a derivative graph to see the slope results clearly. In Graph 7: Optimum pH for Reaction Rate of Peroxidase, the rate of reaction increased drastically from pH 3 with a slope of 0. 00007 to pH 5 with a slope of 0. 0055 and then trails off as the basicness increases at pH 7 with a slope.